Nanoparticle polyanion conjugates and methods of use thereof in detecting analytes

ABSTRACT

This invention provides polyanionic polymer conjugates containing non-nucleotide polyanionic polymers that are useful in detecting target analytes such as proteins or small molecules. The invention also provides nanoparticles bound to polyanionic polymer conjugates and methods of preparation and use thereof. The polyanionic polymer conjugates have the formula: 
       L-O—[PO 2 —O-Z-O] n —PO 2 —O—X         wherein n ranges from 1 to 200; L represents a moiety comprising a functional group for attaching the polyanion polymer to the nanoparticle surface; Z represents a bridging group, and X represents Q, X′ or -Q-X′, wherein Q represents a functional group for attaching a recognition probe to the polyanion polymer, and X′ represents a recognition probe.

RELATED APPLICATIONS

This application claims the benefit of priority from U.S. provisionalapplication No. 60/393,255, filed Jul. 2, 2002, the contents of whichare incorporated herein by reference in their entirety.

FIELD OF INVENTION

This invention relates to polyanionic polymer conjugates containingnon-nucleotide polyanionic polymers (“polyanions”), nanoparticles boundto polyanionic polymer conjugates and methods of preparation and usethereof in detecting target analytes such as proteins or smallmolecules.

BACKGROUND OF THE INVENTION

The binding of polyelectrolytes to gold nanoparticles for stabilizationhas been described (1). When mixing a polyelectrolyte solution with goldnanoparticles, the polymer composition, length, concentration, and totalsalt concentration all influence the coating of particles withpolyelectrolytes (2). More specifically, high salt concentrations(0.05-0.1 M NaCl) have been found to cause aggregation of goldnanoparticles stabilized with polyelectrolytes (1, 2). This represents asignificant limitation to coating nanoparticles with polyelectrolytes(2), and also severely limits the utility of polyanion coatingtechnology since many applications such as biomolecule sensing requirestability to electrolytes as well as temperature. For example, nucleicacid detection is typically performed in buffers that contain salt whichpromote nucleic acid hybridization, and biological fluids (e.g. urine)also contain elevated electrolyte concentrations which would destabilizepolyelectrolyte nanoparticle complexes. Accordingly, a method forbinding polyelectrolytes to nanoparticles that provides conjugates thatare stable to electrolytes as well as temperature, conditions which aretypically found in applications such as biomolecule sensing, would behighly desirable.

SUMMARY OF THE INVENTION

Herein we describe a method for preparing highly stable polyanionnanoparticle conjugates which utilizes a polyanion modified with afunctional group to covalently attach the polyanion to the nanoparticlesurface. This method creates a high density of polyanion moieties on thenanoparticle surface, providing highly stable polyanion-nanoparticleconjugates that may be used in biosensing applications.

This method differs from previous methods that utilize phosphate basednucleic acid moieties (3), which may produce unacceptable background inbiosensing applications, as it is well known in the art that nucleicacids can bind to other nucleic acids, proteins, and small molecules. Inthe invention, the sugar and base moieties that constitute the nucleicacid have been removed, thus reducing or eliminating background inbiosensing applications. This method also differs from previousstrategies that utilize a monolayer of mercaptoundecanoic acid to anchorpolyelectrolyte moieties through electrostatic attraction to the goldnanoparticle surface using a complicated layer-by-layer assemblytechnique (2). In the method described herein, the group responsible forbinding to the nanoparticle surface is directly attached to thepolyanion of interest for binding and the resulting conjugate may beimmobilized on the particle in a single step. In addition, any ligandthat may be used for biomolecule recognition may be attached to thepolymer before or after nanoparticle modification.

The present invention provides polyanionic polymer conjugates containingnon-nucleotide polyanions, nanoparticles bound with polyanionic polymerconjugates and methods for the synthesis of nanoparticles havingpolyanionic polymer conjugates attached thereto, and methods fordetecting target analytes. The polyanions can be any water-soluble andwater stable polymer or co-polymer with a net negative charge and atleast one functional group that is able to bind to the nanoparticlesurface.

More specifically, the invention provides polyanionic polymer conjugatescontaining non-nucleotide polyanions, nanoparticles and methods forcovalent attachment of polyanionic polymers that serve either as“spacer” molecules between the surface of the nanoparticle and therecognition moiety, e.g., a recognition oligonucleotide probe, or as“filler” molecules that cover the surface of the nanoparticle betweenthe oligonucleotide probes. These “spacer” and “filler” arrangements canallow for more efficient binding between the nanoparticle probe andbiomolecule of interest. These nanoparticle probes comprisingpolyanionic polymers and a recogniton element are useful for biomoleculedetection (e.g. nucleic acid sequence or protein), detectingprotein-ligand interactions, separation of a target oligonucleotidesequence from a population of sequences, or other methods as describedpreviously for instance in PCT/US01/10071, filed Mar. 28, 2001 and U.S.Pat. No. 6,361,944, issued Mar. 26, 2002, which are incorporated byreference in their entirety.

Incorporation of the polyanionic polymers into the nanoparticle probesincreases the stability of the nanoparticles in solution, especiallycolloidal gold nanoparticles in high salt solutions. The use ofnon-nucleic acid polyanion polymers in preparing nanoparticle conjugatesis advantageous for nucleic acid and protein detection becausenon-specific binding interference between analytes and nanoparticleconjugate probes can be reduced.

In one embodiment, the invention provides polyanionic polymerconjugates.

In another embodiment, the invention provides nanoparticles havingpolyanionic polymer conjugates attached thereto.

In another embodiment, the invention provides synthetic methods for themanufacture of nanoparticle probes comprising a plurality of polyanionicpolymer conjugates. The polyanionic polymer conjugates may befunctionalized to attach ligands or biomolecules of interest.

In another embodiment, the invention provides for methods ofsynthesizing nanoparticle probes having polyanionic polymers serving asspacer, or linking, molecules between the surface of the nanoparticleand oligonucleotide probe sequences.

In another embodiment, the invention provides for methods ofsynthesizing nanoparticle probes having polyanionic polymers serving asspacer, or linking, molecules between the surface of the nanoparticleand small molecule ligands for detecting or binding biomolecules.

In another embodiment, the invention provides for methods ofsynthesizing nanoparticle probes having polyanionic polymers serving asspacer, or linking, molecules between the surface of the nanoparticleand proteins for detecting or binding biomolecules.

In another embodiment, the invention provides for methods ofsynthesizing nanoparticle probes having polyanionic polymers serving asspacer, or linking, molecules between the surface of the nanoparticleand oligonucleotides for detecting or binding biomolecules.

In another embodiment, the invention provides for methods ofsynthesizing nanoparticle probes having polyanionic polymers serving asspacer, or linking, molecules between the surface of the nanoparticleand carbohydrates for detecting or binding biomolecules.

In another embodiment, the invention provides for methods ofsynthesizing nanoparticle probes having polyanionic polymers serving asfiller molecules on the surface of the nanoparticle, between the probes(e.g. oligonucleotide, protein, etc.) that are also attached to thesurface of the nanoparticle.

In a further embodiment, the invention provides for methods ofsynthesizing nanoparticle probes having polyanionic polymers servingboth as filler and spacer molecules.

These and other embodiments of the invention will become apparent inlight of the detailed description below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is an HPLC chromatogram of polyanion 1 after purification.

FIG. 1B is a UV-visible spectrum of polyanion 1 after purification.

FIG. 2 is a visible absorbance spectrum of gold nanoparticle-polyanion 1conjugates after loading and isolation.

FIG. 3 is a graph depicting a standard curve used to measure the numberof polyanion molecules per gold particle.

FIG. 4A is a graph indicating stability of polyanion 1-gold nanoparticleconjugates at 0.5 M PBS.

FIG. 4B is a graph of stability of citrate modified nanoparticles at 0.5M PBS.

FIG. 5 is a graph indicating stability of polyanion 1-gold nanoparticleconjugates at 0.5 M PBS after heating.

FIG. 6 is an HPLC chromatogram of polyanion 6 after purification.

FIG. 7A shows a reversed phase plate which exhibits spot test resultsfrom solutions of polyanion 6 incubated with Streptavidin or controlsolutions.

FIG. 7B is a UV-vis spectrum of polyanion 6 incubated with Streptavidinor control solutions.

FIG. 8A shows spot test results for free d-biotin/polyanion 6competitive binding experiment for Streptavidin.

FIG. 8B is a UV-visible spectrum of free d-biotin/polyanion 6competitive binding experiment for Streptavidin.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the discovery that non-nucleotidepolyanions may be used to stabilize gold nanoparticles. Thus in oneembodiment, the invention provides polyanionic polymer conjugatescontaining non-nucleotide polyanions. The polyanionic polymer conjugatescontaining non-nucleotide polyanions can be attached to nanoparticles toform nanoparticle probes useful for detecting analytes. The polyanionicpolymer conjugates have the formula:

L-O—[PO₂—O-Z-O]_(n)—PO₂—O—X

wherein n ranges from 1 to 200; L represents a moiety comprising afunctional group for attaching the polyanion polymer to the nanoparticlesurface; Z represents a bridging group, and X represents Q, X′ or -Q-X′,wherein Q represents a functional group for attaching a recognitionprobe to the polyanion polymer, and X′ represents a recognition probe.

As indicated above, L is a moiety comprising a functional group forattaching the polyanionic polymer conjugate to the nanoparticle surface.Examples of suitable functional groups include alkanethiol groups,phosphorothioate groups (see, e.g., U.S. Pat. No. 5,472,881),substituted alkylsiloxanes (see, e.g. Burwell, Chemical Technology, 4,370-377 (1974) and Matteucci and Caruthers, J. Am. Chem. Soc., 103,3185-3191 (1981) for binding of oligonucleotides to silica and glasssurfaces, and Grabar et al., Anal. Chem., 67, 735-743 for binding ofaminoalkylsiloxanes and for similar binding of mercaptoaklylsiloxanes).

The moiety L may also comprise polyfunctional groups such as cyclicdisulfide group, or polythiols or polymers with multiple functionalgroups that can bind to nanoparticles. The cyclic disulfides preferablyhave 5 or 6 atoms in their rings, including the two sulfur atoms.Suitable cyclic disulfides are available commercially or may besynthesized by known procedures. The reduced form of the cyclicdisulfides can also be used. Preferably, a hydrocarbon moiety isattached to the cyclic disulfide. Suitable hydrocarbons are availablecommercially, and are attached to the cyclic disulfides. Preferably thehydrocarbon moiety is a steroid residue. The two sulfur atoms of thecyclic disulfide should preferably be close enough together so that bothof the sulfur atoms can attach simultaneously to the nanoparticle. Mostpreferably, the two sulfur atoms are adjacent to each other. Examples ofcyclic disulfides and polythiols are described in U.S. patentapplication Ser. No. 09/760,500, filed Jan. 12, 2001, and InternationalApplication Number PCT/US01/01190, filed Jan. 12, 2001, which areincorporated herein by reference in their entirety.

As indicated above, Z is a bridging group. As a bridge, Z can be anydesired chemical group. For instance, Z can be a polymer (e.g.,polyethylene glycol, polymethylene), —C₁-C₁₀-alkyl-, —COO—,—CH₂(CH₂)_(v)COO—, —OCO—, R¹N(CH₂)_(v)—NR¹—, —OC(CH₂)_(v)—, —(CH₂)_(v)—,—O—(CH₂)_(v)—O—, —R¹N—(CH₂)_(v)—,

or

v is 0-30 and R′ is H or is G(CH₂)_(v), wherein G is —CH₃, —CHCH₃,—COOH, —CO₂(CH₂)_(v)CH₃, —OH, or —CH₂OH. Preferably, Z is polyethyleneglycol.

X′ represents a recognition probe. By “recognition probe” is meant amolecule containing at least one binding moiety with a binding affinityfor a target analyte. Examples of a recognition probe suitable for usein the invention include, without limitation, a receptor, a nucleotide,a nucleoside, a polynucleotide, an oligonucleotide, double stranded DNA,an antibody, a sugar, a hapten, a protein, a peptide, a nucleic acid, apeptide nucleic acid, an amino acid, a linked nucleic acid, a nucleosidetriphosphate, a carbohydrate, a lipid, a lipid bound protein, anaptamer, a virus, a cell fragment, or a whole cell.

Q, when present, represents a functional group for attaching therecognition probe to the polyanionic polymer conjugate, and can be, forexample, a nucleophile that is naturally present or chemically added tothe polyanion polymer or the recognition probe, such as an amino group,sulfhydryl group, hydroxy group, carboxylate group, or any suitablemoiety. Q may represent —NH, —S—, —O—, or —OOC—.

Nanoparticles useful in the practice of the invention include metal(e.g., gold, silver, copper and platinum), metal oxides (e.g., TiO₂),semiconductor (e.g., CdSe, CdS, and CdS or CdSe coated with ZnS) andmagnetic (e.g., ferromagnetite) colloidal materials. Other nanoparticlesuseful in the practice of the invention include ZnS, ZnO, TiO₂, AgI,AgBr, HgI₂, PbS, PbSe, ZnTe, CdTe, In₂S₃, In₂Se₃, Cd₃P₂, Cd₃As₂, InAs,and GaAs. The size of the nanoparticles is preferably from about 5 nm toabout 150 nm (mean diameter), more preferably from about 5 to about 50nm, most preferably from about 10 to about 30 nm. The nanoparticles mayalso be rods.

Methods of making metal, semiconductor and magnetic nanoparticles arewell-known in the art. See, e.g., Schmid, G. (ed.) Clusters and Colloids(VCH, Weinheim, 1994); Hayat, M. A. (ed.) Colloidal Gold: Principles,Methods, and Applications (Academic Press, San Diego, 1991); Massart,R., IEEE Transactions On Magnetics, 17, 1247 (1981); Ahmadi, T. S. etal., Science, 272, 1924 (1996); Henglein, A. et al., J. Phys. Chem., 99,14129 (1995); Curtis, A. C., et al., Angew. Chem. Int. Ed. Engl., 27,1530 (1988).

Methods of making ZnS, ZnO, TiO₂, AgI, AgBr, HgI₂, PbS, PbSe, ZnTe,CdTe, In₂S₃, In₂Se₃, Cd₃P₂, Cd₃As₂, InAs, and GaAs nanoparticles arealso known in the art. See, e.g., Weller, Angew. Chem. Int. Ed. Engl.,32, 41 (1993); Henglein, Top. Curr. Chem., 143, 113 (1988); Henglein,Chem. Rev., 89, 1861 (1989); Brus, Appl. Phys. A., 53, 465 (1991);Bahncmann, in Photochemical Conversion and Storage of Solar Energy (eds.Pelizetti and Schiavello 1991), page 251; Wang and Herron, J. Phys.Chem., 95, 525 (1991); Olshavsky et al., J. Am. Chem. Soc., 112, 9438(1990); Ushida et al., J. Phys. Chem., 95, 5382 (1992).

Suitable nanoparticles are also commercially available from, e.g., TedPella, Inc. (gold), Amersham Corporation (gold) and Nanoprobes, Inc.(gold).

Presently preferred for use in detecting nucleic acids are goldnanoparticles such as the ones described in International ApplicationNumber PCT/US01/01190, filed Jan. 12, 2001, and U.S. Pat. No. 6,506,564,issued Jan. 14, 2003, which are both incorporated herein by reference intheir entirety. Gold colloidal particles have high extinctioncoefficients for the bands that give rise to their beautiful colors.These intense colors change with particle size, concentration,interparticle distance, and extent of aggregation and shape (geometry)of the aggregates, making these materials particularly attractive forcolorimetric assays. For instance, hybridization of oligonucleotidesattached to gold nanoparticles with oligonucleotides and nucleic acidsresults in an immediate color change visible to the naked eye (see,e.g., the Examples).

In order to bind the polyanionic polymer conjugates to thenanoparticles, the polyanionic polymer conjugates are contacted with thenanoparticles in water for a time sufficient to allow at least some ofthe polyanionic polymer conjugates to bind to the nanoparticles by meansof the functional groups. Such times can be determined empirically. Forinstance, it has been found that a time of about 12-24 hours gives goodresults. Other suitable conditions for binding of the polyanionicpolymer conjugates can also be determined empirically. For instance, aconcentration of about 10-20 nM nanoparticles and incubation at roomtemperature gives good results.

A recognition probe X′ may be attached to a polyanion before thepolyanion is attached to a nanoparticle. In this embodiment, X in thepolyanion conjugate represents either -Q-X′ or X′. Alternatively, arecognition group X′ may be attached to a polyanion after the polyanionhas already been attached to a nanoparticle.

A preferred method for attaching polyanionic polymer conjugates to ananoparticle is based on an aging process described in U.S. applicationSer. Nos. 09/344,667, filed Jun. 25, 1999; 09/603,830, filed Jun. 26,2000; 09/760,500, filed Jan. 12, 2001; 09/820,279, filed Mar. 28, 2001;09/927,777, filed Aug. 10, 2001; and in International application nos.PCT/US97/12783, filed Jul. 21, 1997; PCT/US00/17507, filed Jun. 26,2000; PCT/US01/01190, filed Jan. 12, 2001; PCT/US01/10071, filed Mar.28, 2001, the disclosures of which are incorporated by reference intheir entirety.

The aging process provides nanoparticle probes with enhanced stabilityand selectivity. The polyanionic polymer conjugates are contacted withthe nanoparticles in water for a time sufficient to allow at least someof the polyanionic polymer conjugates to bind to the nanoparticles bymeans of the functional groups. Such times can be determinedempirically. For instance, it has been found that a time of about 12-24hours gives good results. Other suitable conditions for binding of thepolyanionic polymer conjugates can also be determined empirically. Forinstance, a concentration of about 10-20 nM nanoparticles and incubationat room temperature gives good results.

Next, at least one salt is added to the water to form a salt solution.The salt can be any suitable water-soluble salt. For instance, the saltmay be sodium chloride, lithium chloride, potassium chloride, cesiumchloride, ammonium chloride, sodium nitrate, lithium nitrate, cesiumnitrate, sodium acetate, lithium acetate, cesium acetate, ammoniumacetate, a combination of two or more of these salts, or one of thesesalts in phosphate buffer. Preferably, the salt is added as aconcentrated solution, but it could be added as a solid. The salt can beadded to the water all at one time or the salt is added gradually overtime. By “gradually over time” is meant that the salt is added in atleast two portions at intervals spaced apart by a period of time.Suitable time intervals can be determined empirically.

The ionic strength of the salt solution must be sufficient to overcomeat least partially the electrostatic repulsion of the polyanionicpolymer conjugates from each other and, either the electrostaticattraction of the negatively-charged polyanionic polymer conjugates forpositively-charged nanoparticles, or the electrostatic repulsion of thenegatively-charged polyanionic polymer conjugates fromnegatively-charged nanoparticles. Gradually reducing the electrostaticattraction and repulsion by adding the salt gradually over time providesthe highest surface density of polyanionic polymer conjugates on thenanoparticles. Suitable ionic strengths can be determined empiricallyfor each salt or combination of salts. A final concentration of sodiumchloride of from about 0.1 M to about 3.0 M in phosphate buffer,preferably with the concentration of sodium chloride being increasedgradually over time, has been found to give good results.

After adding the salt, the polyanionic polymer conjugates andnanoparticles are incubated in the salt solution for an additionalperiod of time sufficient to allow sufficient additional polyanionicpolymer conjugates to bind to the nanoparticles to produce the stablenanoparticle-polyanion polymer probes. The time of this incubation canbe determined empirically. A total incubation time of about 24-48,preferably 40 hours, gives good results (this is the total time ofincubation; as noted above; the salt concentration can be increasedgradually over this total time). This second period of incubation in thesalt solution is referred to herein as the “aging” step. Other suitableconditions for this “aging” step can also be determined empirically. Forinstance, incubation at room temperature and pH 7.0 gives good results.The solution is then centrifuged and the nanoparticle probes processedas desired.

The probes produced by use of the “aging” step are more stable thanthose produced without the “aging” step. As noted above, this increasedstability is due to the increased density of the polyanionic polymerconjugates on the surfaces of the nanoparticles which is achieved by the“aging” step. The surface density achieved by the “aging” step willdepend on the size and type of nanoparticles and on the length, andconcentration of the polyanionic polymer conjugates. A surface densityadequate to make the nanoparticles stable and the conditions necessaryto obtain it for a desired combination of nanoparticles and polyanionicpolymer conjugates can be determined empirically.

Nanoparticles having polyanionic polymer conjugates of the inventionattached thereto (referred to herein as “nanoparticle probes” or“nanoparticle conjugates”) have a variety of uses. For instance, theycan be used as probes to detect or quantitate analytes. See, e.g., PCTapplication WO 98/04740; PCT application WO 98/21587; Storhoff et al.,J. Clust. Sci., 8: 179 (1997); Brousseau et al., J. Am. Chem. Soc., 120:7645 (1998); Freeman et al., Science, 267: 1629 (1995); Zhu et al., J.Am. Chem. Soc., 119: 235 (1997); Mirkin et al., Nature, 382: 607 (1996);Elghanian et al., Science, 277: 1078 (1997); Storhoff et al., J. Am.Chem. Soc., 120: 1959 (1998). Analytes that can be detected orquantitated according to the invention include polysaccharides, lipids,lipopolysaccharides, proteins, glycoproteins, lipoproteins,nucleoproteins, peptides, oligonucleotides, and nucleic acids. Specificanalytes include antibodies, immunoglobulins, albumin, hemoglobin,coagulation factors, peptide and protein hormones (e.g., insulin,gonadotropin, somatotropin), non-peptide hormones, interleukins,interferons, other cytokines, peptides comprising a tumor-specificepitope (i.e., an epitope found only on a tumor-specific protein), cells(e.g., red blood cells), cell surface molecules (e.g., CD antigens,integrins, cell receptors), microorganisms (viruses, bacteria,parasites, molds, and fungi), fragments, portions, components orproducts of microorganisms, small organic molecules (e.g., digoxin,heroin, cocaine, morphine, mescaline, lysergic acid,tetrahydrocannabinol, cannabinol, steroids, pentamidine, and biotin),etc. Nucleic acids and oligonucleotides that can be detected orquantitated include genes (e.g., a gene associated with a particulardisease), viral RNA and DNA, bacterial DNA, fungal DNA, mammalian DNA(e.g., human DNA), cDNA, mRNA, RNA and DNA fragments, oligonucleotides,synthetic oligonucleotides, modified oligonucleotides, single-strandedand double-stranded nucleic acids, natural and synthetic nucleic acids,etc.

To serve as probes, the nanoparticle probes of the invention include therecognition probe section X′, as part of the polyanionic polymerconjugates attached thereto, which allows the nanoparticle probes tobind specifically to the analyte. Suitable recognition probes X′ andmethods of making them are well known in the art. For instance,essentially any analyte can be detected or quantitated using antibodiesspecific for the analyte. In addition, any molecule which bindsspecifically to the analyte can be used, and many such molecules areknown in the art. For instance, nucleic acids can be detected orquantitated using oligonucleotides having a sequence which iscomplementary to at least a portion of the analyte nucleic acid. Also,lectins can be used to detect or quantitate polysaccharides andglycosylated proteins. As another example, a receptor can be used todetect its ligand and vice versa. Many other suitable recognition probesX′ are known.

To perform an assay according to the invention, a sample suspected ofcontaining an analyte is contacted with nanoparticle probes havingrecognition probes X′ attached thereto. Any type of sample can be used.For instance, the sample may be a biological fluid (e.g., serum, plasma,blood, saliva, and urine), cells, cell lysates, tissues, libraries ofcompounds (e.g., organic chemicals or peptides), solutions containingPCR components, etc. Conditions and formats for performing such assaysare well known in the art (see, e.g., the references cited above) or canbe determined empirically by those of ordinary skill in the art.Finally, the property or properties of the nanoparticles is (are)detected or measured in order to detect or quantitate the analyte.Preferably, the property is redox activity or optical activity (e.g.,fluorescence or color as described below). Methods of detecting andmeasuring these properties are well known in the art.

One example of a method for detecting a target analyte wherein thetarget analyte is a nucleic acid comprises contacting the nucleic acidwith one or more types of nanoparticle probes of the invention. Thenucleic acid to be detected has at least two portions. The lengths ofthese portions and the distance(s), if any, between them are chosen sothat when the recognition probes on the nanoparticle probes hybridize tothe nucleic acid, a detectable change occurs. These lengths anddistances can be determined empirically and will depend on the type ofparticle used and its size and the type of electrolyte which will bepresent in solutions used in the assay (as is known in the art, certainelectrolytes affect the conformation of nucleic acids).

Also, when a nucleic acid is to be detected in the presence of othernucleic acids, the portions of the nucleic acid to which the recognitionprobes on the nanoparticle probes are to bind must be chosen so thatthey contain sufficient unique sequence so that detection of the nucleicacid will be specific. Guidelines for doing so are well known in theart.

Although nucleic acids may contain repeating sequences close enough toeach other so that only one type of nanoparticle probe need be used,this will be a rare occurrence. In general, the chosen portions of thenucleic acid will have different sequences and will be contacted withnanoparticles having polyanionic polymer conjugates attached thereto,wherein the conjugates carry two or more different recognition probes,preferably attached to different nanoparticles. Additional portions ofthe DNA could be targeted with corresponding nanoparticles. Targetingseveral portions of a nucleic acid increases the magnitude of thedetectable change.

The contacting of the nanoparticle probes with the nucleic acid takesplace under conditions effective for hybridization of the recognitionprobes on the polyanionic polymer conjugates attached to thenanoparticles with the target sequence(s) of the nucleic acid. Thesehybridization conditions are well known in the art and can readily beoptimized for the particular system employed. See, e.g., Sambrook etal., Molecular Cloning: A Laboratory Manual (2nd ed. 1989). Preferablystringent hybridization conditions are employed.

Faster hybridization can be obtained by freezing and thawing a solutioncontaining the nucleic acid to be detected and the nanoparticle probes.The solution may be frozen in any convenient manner, such as placing itin a dry ice-alcohol bath for a sufficient time for the solution tofreeze (generally about 1 minute for 100 microliters of solution). Thesolution must be thawed at a temperature below the thermal denaturationtemperature, which can conveniently be room temperature for mostcombinations of nanoparticle probes and nucleic acids. The hybridizationis complete, and the detectable change may be observed, after thawingthe solution.

The rate of hybridization can also be increased by warming the solutioncontaining the nucleic acid to be detected and the nanoparticle probesto a temperature below the dissociation temperature (Tm) for the complexformed between the recognition probes on the nanoparticle probes and thetarget nucleic acid. Alternatively, rapid hybridization can be achievedby heating above the dissociation temperature (Tm) and allowing thesolution to cool.

The rate of hybridization can also be increased by increasing the saltconcentration (e.g., from 0.1 M to 1 M NaCl). The rate of the reactioncan also be increased by adding a volume exclusion reagent such asdextran sulfate.

The detectable change that occurs upon hybridization of the recognitionprobes on the nanoparticle probes to the nucleic acid may be an opticalchange (e.g. color change), the formation of aggregates of thenanoparticles, or the precipitation of the aggregated nanoparticles. Theoptical changes can be observed with the naked eye or spectroscopically.The formation of aggregates of the nanoparticles can be observed byelectron microscopy or by nephelometry. The precipitation of theaggregated nanoparticles can be observed with the naked eye ormicroscopically. Preferred are color changes observable with the nakedeye.

The observation of a color change with the naked eye can be made morereadily against a background of a contrasting color. For instance, whengold nanoparticles are used, the observation of a color change isfacilitated by spotting a sample of the hybridization solution on asolid white surface (such as silica or alumina TLC plates, filter paper,cellulose nitrate membranes, and nylon membranes, preferably a nylonmembrane) and allowing the spot to dry. Initially, the spot retains thecolor of the hybridization solution (which ranges from pink/red, in theabsence of hybridization, to purplish-red/purple, if there has beenhybridization). On drying at room temperature or 80° C. (temperature isnot critical), a blue spot develops if the polyanionpolymer-nanoparticle conjugates had been linked by hybridization withthe target nucleic acid prior to spotting. In the absence ofhybridization (e.g., because no target nucleic acid is present), thespot is pink. The blue and the pink spots are stable and do not changeon subsequent cooling or heating or over time. They provide a convenientpermanent record of the test. No other steps (such as a separation ofhybridized and unhybridized nanoparticle probes) are necessary toobserve the color change. The color change may be quantitated byrecording the plate image with an optical scanning device such as aflatbed scanner or CCD camera, and analyzing the amount and type ofcolor of each individual spot. Alternatively, a color filter (e.g. redfilter) may be used to filter out specific colors so that the signalintensity of each spot may be recorded and analyzed.

An alternate method for easily visualizing the assay results is to spota sample of nanoparticle probes hybridized to a target nucleic acid on aglass fiber filter (e.g., Borosilicate Microfiber Filter, 0.7 micronpore size, grade FG75, for use with gold nanoparticles 13 nm in size),while drawing the liquid through the filter. Subsequent rinsing withwater washes the excess, non-hybridized nanoparticle probes through thefilter, leaving behind an observable spot comprising the aggregatesgenerated by hybridization of the recognition probes on the nanoparticleprobes with the target nucleic acid (retained because these aggregatesare larger than the pores of the filter). This technique may provide forgreater sensitivity, since an excess of nanoparticle probes can be used.

Binding may also be detected by light scattering techniques such asthose described in U.S. provisional application No. 60/474,569, filedMay 30, 2003, which is incorporated herein by reference in its entirety.In such methods, binding analytes, e.g., nucleic acids or proteins, canbe detected through light scattering techniques, where a change in lightscattering caused by the formation of nanoparticle label complexeswithin the penetration depth of an evanescent wave of a wave guidesignals the presence of analyte.

Some embodiments of the method of detecting nucleic acid utilize asubstrate. By employing a substrate, the detectable change (the signal)can be amplified and the sensitivity of the assay increased.

Any substrate can be used which allows observation of the detectablechange. Suitable substrates include transparent solid surfaces (e.g.,glass, quartz, plastics and other polymers), opaque solid surface (e.g.,white solid surfaces, such as TLC silica plates, filter paper, glassfiber filters, cellulose nitrate membranes, nylon membranes), andconducting solid surfaces (e.g., indium-tin-oxide (ITO)). The substratecan be any shape or thickness, but generally will be flat and thin.Preferred are transparent substrates such as glass (e.g., glass slides)or plastics (e.g., wells of microtiter plates).

In one embodiment of the method of detecting nucleic acid using asubstrate, oligonucleotides are attached to the substrate. Theoligonucleotides can be attached to the substrates as described in,e.g., Chrisey et al., Nucleic Acids Res., 24, 3031-3039 (1996); Chriseyet al., Nucleic Acids Res., 24, 3040-3047 (1996); Mucic et al., Chem.Commun., 555 (1996); Zimmermann and Cox, Nucleic Acids Res., 22, 492(1994); Bottomley et al., J. Vac. Sci. Technol. A, 10, 591 (1992); andHegner et al., FEBS Lett., 336, 452 (1993).

The oligonucleotides attached to the substrate have a sequencecomplementary to a first portion of the sequence of a nucleic acid to bedetected. The nucleic acid is contacted with the substrate underconditions effective to allow hybridization of the oligonucleotides onthe substrate with the nucleic acid. In this manner the nucleic acidbecomes bound to the substrate. Any unbound nucleic acid is preferablywashed from the substrate before adding nanoparticle probes.

Next, the nucleic acid bound to the substrate is contacted with a firsttype of nanoparticle probes. The recognition probes on the nanoparticleprobes have a sequence complementary to a second portion of the sequenceof the nucleic acid, and the contacting takes place under conditionseffective to allow hybridization of the recognition probes on thenanoparticles with the nucleic acid. In this manner the first type ofnanoparticle probes become bound to the substrate. After thenanoparticle probes are bound to the substrate, the substrate is washedto remove any unbound nanoparticle probes and nucleic acid.

The recognition probes on the first type of nanoparticle probes may allhave the same sequence or may have different sequences that hybridizewith different portions of the nucleic acid to be detected. Whenrecognition probes having different sequences are used, eachnanoparticle may have all of the different recognition probes attachedto it or, preferably, the different recognition probes are attached todifferent nanoparticles. Alternatively, the recognition probes on eachof the first type of nanoparticle probes may have a plurality ofdifferent sequences, at least one of which must hybridize with a portionof the nucleic acid to be detected.

The first type of nanoparticle probes bound to the substrate areoptionally contacted with a second type of nanoparticle probes. Theserecognition probes (on the second type of nanoparticle probes) have asequence complementary to at least a portion of the sequence(s) of therecognition probes on the first type nanoparticle probes, and thecontacting takes place under conditions effective to allow hybridizationof the recognition probes on the first type of nanoparticle probes withthose on the second type of nanoparticle probes. After the nanoparticlesare bound, the substrate is preferably washed to remove any unboundconjugates.

The combination of hybridizations produces a detectable change. Thedetectable changes are the same as those described above, except thatthe use of the second type of nanoparticle probes provides multiplehybridizations which result in an amplification of the detectablechange. In particular, since each of the first type of nanoparticleprobes has multiple recognition probes (having the same or differentsequences) attached to it, each of the first type of nanoparticle probescan hybridize to a plurality of the second type of nanoparticle probes.Also, the first type of nanoparticle probes may be hybridized to morethan one portion of the nucleic acid to be detected. The amplificationprovided by the multiple hybridizations may make the change detectablefor the first time or may increase the magnitude of the detectablechange. This amplification increases the sensitivity of the assay,allowing for detection of small amounts of nucleic acid.

If desired, additional layers of nanoparticles can be built up bysuccessive additions of the first and second types of nanoparticleprobes. In this way, the number of nanoparticles immobilized permolecule of target nucleic acid can be further increased with acorresponding increase in intensity of the signal.

In one embodiment for detection of non-nucleic acid analytes (see forexample U.S. patent application Ser. No. 09/820,279, filed Mar. 28,2001, and International application PCT/01/10071, filed Mar. 28, 2001,each of which is incorporated herein by reference) the analyte may bebound directly or indirectly, via covalent or non-covalent interactions,to a substrate. The substrates are the same type as described above. Forindirect binding, the analyte can be bound to the substrate via alinker, e.g., an oligonucleotide or other spacer molecule.Alternatively, the analyte may be modified by binding it to anoligonucleotide having a sequence that is complementary to at least aportion of the sequence of a capture probe, such as an oligonucleotide,bound to a substrate. The nanoparticle probes having recognition probesthat can hybridize to the analyte are then contacted with the substrateunder conditions effective to allow the specific binding of therecognition probes to the analyte bound to the substrate and thepresence of the analyte can be visually detected either by formation ofa spot on the substrate or through the use of staining material such assilver on gold stain.

In another method for detecting analytes, the target analyte can bemodified by attaching the analyte to a polyanionic polymer conjugate asthe recognition probe and attaching the polyanionic polymer conjugate toa nanoparticle. Thereafter, the modified nanoparticle probe is contactedwith a substrate having a second member of the recognition couple boundthereto. The presence of the analyte can be visually detected either byformation of a spot on the substrate or through the use of stainingmaterial such as silver on gold stain.

In yet another method for detecting analytes, the target analyte ismodified by binding it to an oligonucleotide having a sequence that iscomplementary to at least a portion of a sequence of a recognition probeon a nanoparticle probe. The modified target is then coupled to therecognition probe on the nanoparticle by contacting the modified targetand the nanoparticle under conditions effective for hybridizationbetween the oligonucleotide bound to the target and the recognitionprobe on the nanoparticle. The hybridized complex is then contacted witha substrate having a recognition group for the analyte bound thereto.The presence of the analyte can be visually detected either by formationof a spot on the substrate or through the use of staining material suchas silver on gold stain.

When a substrate is employed, a detectable change can be produced orenhanced by staining. Staining material, e.g., gold, silver, etc., canbe used to produce or enhance a detectable change in any assay performedon a substrate, including those described above. For instance, silverstaining can be employed with any type of nanoparticles that catalyzethe reduction of silver. Preferred are nanoparticles made of noblemetals (e.g., gold and silver). See Bassell, et al., J. Cell Biol., 126,863-876 (1994); Braun-Howland et al., Biotechniques, 13, 928-931 (1992).If the nanoparticles being employed for the detection of analyte do notcatalyze the reduction of silver, then silver ions can be complexed tothe target analyte to catalyze the reduction. See Braun et al., Nature,391, 775 (1998). Also, silver stains are known which can react with thephosphate groups on nucleic acids.

The invention further provides kits for detecting the presence orabsence of a target analyte in a sample comprising. A kit may comprise acontainer holding polyanionic polymer conjugates having recognitionprobes attached thereto. A kit may also comprise a container holdingpolyanionic polymer conjugates having recognition probes, wherein thepolyanionic polymer conjugates are attached to nanoparticles. The kitsmay also contain other reagents and items useful for performing theassays. The reagents may include controls, standards, PCR reagents,hybridization reagents, buffers, etc. Other items which be provided aspart of the kit include reaction devices (e.g., test tubes, microtiterplates, solid surfaces (possibly having a capture molecule attachedthereto), syringes, pipettes, cuvettes, containers, etc.

The following examples are illustrative of the invention but do notserve to limit its scope.

EXAMPLES

To illustrate the invention a representative non-nucleotide basedpolyanionic polymer conjugate was prepared, Scheme 1. The conjugatecontains three parts: 1) a linker such as a steroid cyclic disulfideanchor that is used as a linker for the gold nanoparticle surface,⁷ 2) aphosphate based polyanion backbone with a bridging group (e.g.polyethylene glycol) designed to provide nanoparticle stabilization, and3) a fluorescein label as a tag for purification and quantitation.

a. Preparation and Purification of a Polyanion Polymer

The molecule was prepared using standard phosphoramidite chemistry on anExpedite 8909 synthesizer and purified by reverse phase HPLC usingstandard conditions⁵ while monitoring the 494 nm absorption maximum ofthe fluorescein tag. The purified product exhibited a doublet atretention times of 37.8 and 38.2 minutes respectively when monitored at494 nm indicating the presence of both the steroid cyclic disulfideanchor and the fluorescein tag, FIG. 1. The retention time and thedoublet observed for the product are consistent with oligonucleotidesmodified with the steroid cyclic disulfide linker, and the absorbance at494 nm is characteristic for the fluorescein chromophore. The purifiedpolyanion product was quantitated using the absorbance at 494 nm(ε₄₉₄=75000 M⁻¹ cm⁻¹).

b. Loading onto Nanoparticles

The polyanion was loaded onto ˜16.5 nm diameter gold nanoparticlesprepared via the citrate method⁶ using the following procedure. The goldnanoparticle solution (2 mL, ˜14 nM) was mixed with polyanion DNA (finalconcentration=3.6 uM) and buffered to pH 7 at 10 mM phosphate (pH 7) andincubated at room temperature overnight. The salt concentration wassubsequently adjusted to 0.1 M phosphate buffered saline (PBS; 10 mMphosphate, 0.1 M NaCl, pH 7) using 4 M PBS (10 mM phosphate, 4 M NaCl,pH 7), and the solution was incubated for >40 hours. The probes wereisolated via centrifugation (13000 rpm×25 minutes), washed once withwater, and redispersed to a final gold nanoparticle concentration of 10nM using 10 mM phosphate (pH 7) buffer, 0.01% azide. The polyanioncoated gold nanoparticles were stable to centrifugation and 0.1 M PBS asevidenced by the UV-vis spectrum after isolation which exhibits anabsorption maximum of 524 nm which is characteristic of DNA-modifiedgold nanoparticles prepared by the same procedure, FIG. 2.⁵

c. Quantitation of Polyanion Conjugates Attached to the GoldNanoparticles

The polyanion conjugates attached to the gold nanoparticles werequantitated using the absorbance signature associated with thefluorescein to demonstrate that the polyanion conjugates were attachedto the nanoparticles. To quantitate the fluorescein signature, the goldparticles were dissolved to remove the absorbance associated with theparticles using the following procedure. The polyanion conjugated goldnanoparticles (200 ul, 10 nM) were mixed with 0.1 M I₂ (10 ul) andincubated at room temperature for 10 minutes which dissolved the goldnanoparticles turning the solution a yellowish color. A solution of 0.1M sodium thiosulfate (20 uL) was added to reduce the iodine andincubated for five minutes at room temperature. The absorbance of thissolution was measured at 516 mm (absorption maxima of fluorescein asmodified by treatment with I₂/thiosulfate) without dilution toquantitate the amount of flourescein present and compared to a standardconcentration curve of the polyanion (without gold nanoparticle)prepared using the same procedure, FIG. 3. The standard curve for theconcentration range of interest was linear. Using the equation from thebest fit line, the polyanion concentration from the gold probe solutionwas measured to be 1.51 uM (Absorbance at 516 nms=0.17 AU). From thestarting ˜10 nM probe solution, this equates to ˜151 polyanion moleculesper particle.

d. Stability of Polyanion Conjugates Attached to the Gold Nanoparticles

Stability towards elevated electrolyte concentrations as well astemperature is critical for biomolecule assays which are typicallyperformed under such conditions. The robustness of the polyanion goldconjugates as compared to the citrate stabilized gold nanoparticles wastested by first monitoring the stability of the two solutions at 0.5 MPBS over a four hour time period, FIG. 4. In this assay, the polyanionprobe solution (5 nM, 100 uL) was mixed with 100 ul of 1 M PBS (0.5 MPBS final concentration) at room temperature, and the UV-vis spectrumwas monitored as a function of time. This process was repeated for thecitrate modified gold nanoparticles adjusted to 5 nM particleconcentration in 10 mM phosphate pH 7. The polyanion gold conjugatesexhibit little loss in signal over the four hour time period and nodetectable shifts in the UV-visible spectrum. By contrast, the citratemodified gold nanoparticles exhibit a red shift within 15 seconds ofmixing indicating instability followed by almost total loss of signalover the four hour time period. This data clearly demonstrates that thepolyanion conjugated gold nanoparticles exhibit significantly enhancedstability when compared to the citrate modified gold nanoparticles.These studies therefore provide data that indicate that the polyanionstabilized gold nanoparticles may be used under conditions typicallyemployed for detection of biomolecules such as DNA and at goldnanoparticle concentrations that may be detected optically or via othermethods.⁵ This enhanced stability clearly distinguishes the polyaniongold probes described here from previously prepared polyanion gold probeconjugates.^(1,2)

The thermal stability of the polyanion gold nanoparticle conjugates(prepared in 0.5 M PBS as described in FIG. 4 assay) was tested byheating the solution to 95° C. for five minutes and 10 minutes andcomparing the resulting UV-visible spectrum to the same solution thathad not undergone heat treatment, FIG. 5. Under these conditions, thepolyanion gold nanoparticle conjugates are stable for five minutes at95° C. as little change in the UV-visible spectrum was observed. Afterheating for 10 minutes, a slight red shift in the UV-visible spectrumwas observed indicating slight instability to prolonged heating.Nonetheless, these data demonstrate that the polyanion gold nanoparticleconjugates are stable to extreme temperatures for short periods of time(<five minutes).

e. Detection of Biomolecules

After demonstrating that polyanions were good stabilizers for goldnanoparticles, the next goal was to demonstrate utility of the polyanionprobes in the detection of biomolecules. The biotin/Streptavidin bindingpair was chosen as a model system to demonstrate this principle due tothe high affinity of the binding and the multiple binding sitesassociated with Streptavidin. Polyanion sequence 6 was synthesized usingstandard phosphoramidite chemistry with a Biotin-TEG CPG support forbiotin incorporation, Scheme 2. This Polyanion can be segmented intofour distinct portions 1) the steroid disulfide anchor⁷, 2) aNitroindole UV tag used for purification and quantification, 3) aphosphate-based polyanion backbone (e.g., a polyethylene glycol basedspacer), and 4) the tetraethylene glycol Biotin complex used forStreptavidin recognition.

Reverse phase HPLC was used to purify polyanion 6 using the sameprotocol as for Polyanion 1 while monitoring the absorption at 265 nmand 328 nm, characteristic absorbance maxima of the nitroindole tag.Polyanion 6 exhibited a doublet with retention times of 39.8 and 40.1minutes at both monitored wavelengths indicating the presence of thesteroid disulfide linker and the nitroindole tag, FIG. 6.

Polyanion 6 was conjugated to ˜30 nm diameter gold particles purchasedfrom BBI, Inc (BBI measured mean diameter=30.9 nm by TEM). The initialsolution of gold colloid (0.33 nM) was mixed with Polyanion 6 (finalconcentration=800 nM) and incubated for 64 hrs at room temperature. Theprobes were isolated from solution by centrifugation (5,000 rpm for 20minutes), washed once in water and redispersed to 1.7 nM gold particleconcentration in 10 mM Phosphate (pH 7) buffer, 0.01% Azide.

In previous experiments, it has been demonstrated that DNA-modified goldnanoparticle probes may be used to detect biomolecules such as DNA insolution by monitoring color changes associated with particleaggregation either in solution or by depositing aliquots of the solutiononto a reverse phase plate and drying, which is referred to as the spottest.⁵ To demonstrate that polyanion gold nanoparticle conjugates may beused for biomolecule detection, a streptavidin binding experiment wasperformed using the polyanion 6 conjugated gold nanoparticle probeswhich contain biotin functionalities. In a typical experiment, theprobes (24 μl, 1.1 nM) were combined with one of the following reagents(3 μl): water, Bovine Serum Albumin (4 μg/ml), or Streptavidin (1 μg/ml,10 μg/ml), and diluted to 0.055 M NaCl using 20 ul of 1×PBS (0.15 MSodium Chloride) and 7 ul of water. After a fifteen minute incubation atroom temperature, a 3 ul aliquot of the solution was spotted onto areverse phase plate and dried to monitor color changes associated withparticle aggregation, FIG. 7.A.⁵ Both solutions containing theStreptavidin exhibited a colorimetric transition from red to purple orblue which indicates Streptavidin induced particle aggregation. Thecontrol solutions containing the protein BSA or no target exhibited ared color when spotted indicating that no significant aggregation hadtaken place. The colorimetric changes associated with the Streptavidininduced polyanion probe aggregation could also be monitored byUV-visible spectroscopy, FIG. 7.B The UV-vis data clearly shows thecharacteristic red shift associated with particle aggregation in thepresence of Streptavidin, but not in the presence of the control proteinBSA.

To further demonstrate the specificity of the biotinylated goldprobe/Streptavidin reaction, a competitive binding experiment wasperformed where the Streptavidin was preincubated with free d-Biotinprior to probe incubation. In a typical experiment, Streptavidinsolutions (3 ul of 1 μg/ml) were prepared with a dilution series of freed-Biotin (4.6 μl of 900 μM, 90 μM and 9 μM) and diluted with water (10μl total volume). These solutions were incubated at room temperature for10 minutes then added to a solution of Polyanion 6 (24 μl of 1.1 nM goldprobes) and buffered with 1×PBS (20 μl of 0.15 M Sodium Chloride). Aftera fifteen minute incubation, a 3 ul aliquot of the sample was placed ona reverse phase TLC plate and allowed to dry, FIG. 8.A. The remainder ofthe solution was diluted in phosphate buffer (0.1 M Sodium Phosphate pH7 to 200 μl total volume), and a UV-vis spectrum was recorded, FIG. 8.B.Both the spotted samples and the UV-Vis spectra exhibit a trend ofdecreasing calorimetric red shifts as the free d-Biotin concentration isincreased, with the solution containing the highest concentration offree d-Biotin exhibiting a similar color and UV-vis spectrum whencompared to the BSA and no target controls. This trend indicates anincreasing inhibition of binding of the biotinylated probes toStreptavidin as the free d-Biotin concentration is increased whichresults in inhibition of particle aggregation. In conclusion, theseexperiments clearly demonstrate that functionalized polyanion probes maybe used in biomolecule detection schemes based on monitoring a signatureassociated with the nanoparticle probes.

Experimental, Preparation and Characterization of Polyanion ConjugatesReagents and General Methods

Spacer phosphoramidite 9, 3′-(6-FAM) CPG, 5-Nitroindole CEphosphoramidite, and Biotin TEG CPG were purchased from Glen Research,Inc (Sterling, Va.). The “steroid-disulfide” phosphoramidite wasprepared according to methods developed by Letsinger and coworkers.⁷16.5 nm diameter gold nanoparticles were prepared via the citratemethod.⁶ 30.9 nm diameter gold nanoparticles were purchased fromBBInternational (Cardiff, UK).

Polyanion synthesis was performed using an Applied Biosystems 8909nucleic acid synthesizer. High performance liquid chromatography (HPLC)of the polyanions was performed using an Agilent series 1100 HPLC.Electronic absorption spectra were recorded using an Agilent 8453 diodearray spectrophotometer. Nanoparticle solutions were centrifuged using aBeckman Coulter Microfuge 18 centrifuge.

Protocols

The polyanion molecules were prepared using standard phospharamidtechemistry.⁷ Polyanion 1 was synthesized on a 1 umol scale using a3′-(6-FAM) CPG support. Polyanion 6 was synthesized on a 1 umol scaleusing a Biotin TEG CPG support. After synthesis, the CPG-boundpolyanions were cleaved from the solid support in 1 mL of concentratedammonium hydroxide at 55° C. for 14 hours. The ammonia was subsequentlyblown off using a stream of nitrogen. An HPLC equipped with areverse-phase Vydac preparative column (5 um, 250×10 mm) was used forpolyanion purification. Purification was performed using 0.03 Mtriethylammonium acetate (TEAA), pH 7 buffer with a 1%/min. gradient of95% CH₃CN/5% 0.03 M TEAA at a flow rate of 4 mL/min. After purificationthe buffer was evaporated, and the polyanions were resuspended in 25 mMphosphate buffer (pH 7). An analytical HPLC was performed afterpurification to assess the resulting purity of the polyanions using thesame buffer system with a reverse phase Vydac analytical column (5 um,250×4 mm) at a flow rate of 1 mL/min. The polyanion-gold nanoparticleconjugates were prepared as described in the report section.

REFERENCES

-   (1) M. A. Hayat, Ed., Colloidal Gold: Principles, Methods, and    Applications, vol. 1 (Academic Press, San Diego, 1989).-   (2) D. I. Gittins, F. Caruso, J. Phys. Chem. B 105, 6846-6852    (2001).-   (3) C. A. Mirkin, R. L. Letsinger, R. C. Mucic, J. J. Storhoff,    Nature 382, 607-609 (Aug. 15, 1996).-   (4) Letsinger, R. L.; Elghanian, R.; Viswanadham, G.; Mirkin, C. A.    Bioconjugate Chemistry 2000, 11, 289-291.-   (5) Storhoff, J. J.; Elghanian, R.; Mucic, R. C.; Mirkin, C. A.;    Letsinger, R. L. Journal of the American Chemical Society 1998, 120,    1959-1964.-   (6) Grabar, K. C.; Freeman, R. G.; Hommer, M. B.; Natan, M. J.    Analytical Chemistry 1995, 67, 735-743.-   (7) Oligonucleotides and Analogues, 1^(st) ed.; Eckstein, F., Ed.;    Oxford University Press: New York, 1991.

1-9. (canceled)
 10. A nanoparticle having a plurality of polyanionicpolymer conjugates attached thereto, said polyanionic polymer conjugateshaving the formula L-O—[PO₂—O-Z-O]_(n)—PO₂—O—X wherein n ranges from 1to 200; L represents a moiety comprising a functional group forattaching the polyanion polymer to a nanoparticle surface wherein thepolyanion polymer is a non-nucleotide Polyanion polymer; Z represents abridging group, and X represents Q, X′ or -Q-X′, wherein Q represents afunctional group for attaching a recognition probe to the polyanionpolymer, and X′ represents a recognition probe.
 11. The nanoparticle ofclaim 10, wherein the polyanionic polymer conjugate further comprises adetection label bound thereto.
 12. The nanoparticle of claim 11, whereinthe detection label comprises a chromophore, a fluorescent label, a UVlabel, a radioisotope, a Raman label or a SERS (surface enhanced ramanspectroscopy) label, or an enzyme.
 13. The nanoparticle of claim 10,wherein the functional group for attaching a recognition probe to thepolyanion polymer comprises a carboxylic acid or an amino group.
 14. Thenanoparticle of claim 10, wherein the recognition probe comprises aprotein, a peptide, a nucleic acid, a peptide nucleic acid, a linkednucleic acid, a nucleoside triphosphate, a carbohydrate, a lipid, alipid bound protein, an aptamer, a virus, a cell fragment, or a wholecell.
 15. The nanoparticle of claim 14, wherein the lipid bound proteincomprises a G-protein coupled receptor.
 16. The nanoparticle of claim10, wherein the recognition probe comprises an antibody, an antigen, areceptor, or a ligand.
 17. The nanoparticle of claim 10 wherein Lcomprises an alkanethiol containing group, a phosphorothioate containinggroup, a substituted alkylsiloxane containing group, a polythiolcontaining group, or a cyclic disulfide containing group.
 18. Thenanoparticle of claim 10 wherein Z comprises a polymer, —C₁-C₁₀-alkyl-,—COO—, —CH₂(CH₂)_(v)COO—, —OCO—, R¹N(CH₂)_(v)—NR¹—, —OC(CH₂)_(v)—,—(CH₂)_(v)—, —O—(CH₂)_(v)—O—, —R¹N—(CH₂)_(v)—,

or

v is 0-30 and R¹ is H or is G(CH₂)_(v), wherein G is —CH₃, —CHCH₃,—COOH, —CO₂(CH₂)_(v)CH₃, —OH, or —CH₂OH. 19-42. (canceled)
 43. A kit fordetecting the presence or absence of a target analyte in a samplecomprising: (a) nanoparticles having polyanionic polymer conjugatesbound thereto, wherein the polyanion polymers have the formula:L-O—[PO₂—O-Z-O]_(n)—PO₂—O—X wherein n ranges from 1 to 200; L representsa moiety comprising a functional group for attaching the polyanionpolymer to a nanoparticle surface wherein the polyanion polymer is anon-nucleotide polyanion polymer; Z represents a bridging group, and Xrepresents Q, X′ or -Q-X′, wherein Q represents a functional group forattaching a recognition probe to the polyanion polymer, and X′represents a recognition probe; and (b) an optional substrate forobserving a detectable change.
 44. The kit of claim 43, wherein thepolyanionic polymer conjugate further comprises a detection label boundthereto.
 45. The kit of claim 44, wherein the detection label comprisesa chromophore, a fluorescent label, a UV label, a radioisotope, a Ramanlabel or a SERS (surface enhanced raman spectroscopy) label, or anenzyme.
 46. The kit of claim 43 wherein the functional group forattaching a recognition probe to the polyanion polymer comprises acarboxylic acid or an amino group.
 47. The kit of claim 43, wherein therecognition probe comprises a protein, a peptide, a nucleic acid, apeptide nucleic acid, a linked nucleic acid, a nucleoside triphosphate,a carbohydrate, a lipid, a lipid bound protein, an aptamer, a virus, acell fragment, or a whole cell.
 48. The kit of claim 47, wherein thelipid bound protein comprises a G-protein coupled receptor.
 49. The kitof claim 43, wherein the recognition probe comprises an antibody, anantigen, a receptor, or a ligand.
 50. The kit of claim 43 wherein thesubstrate is a transparent substrate or an opaque white substrate. 51.The kit of claim 43 wherein L comprises an alkanethiol containing group,a phosphorothioate containing group, a substituted alkylsiloxanecontaining group, a polythiol containing group, or a cyclic disulfidecontaining group.
 52. The kit of claim 43 wherein Z comprises a polymer,—C₁-C₁₀-alkyl-, —COO—, —CH₂(CH₂)_(v)COO—, —OCO—, R¹N(CH₂)_(v)—NR¹—,—OC(CH₂)_(v)—, —(CH₂)_(v)—, —O—(CH₂)_(v)—O—, —R¹N—(CH₂)_(v)—,

or

v is 0-30 and R¹ is H or is G(CH₂)_(v), wherein G is —CH₃, —CHCH₃,—COOH, —CO₂(CH₂)_(v)CH₃, —OH, or —CH₂OH.